1. Field of the Invention
This invention relates to a method for increasing the ability of apoglucose oxidase to combine with flavin adenine dinucleotide (FAD) and FAD derivatives to form active glucose oxidase, i.e., a method for activating apoglucose oxidase. The invention further relates to homogeneous specific binding assay methods, reagent means, and test kits for determining a ligand, such as an antigen, hapten or antibody, in a liquid medium wherein FAD is employed as label and wherein the FAD label is monitored by its ability to combine with apoglucose oxidase to form active glucose oxidase.
Glucose oxidase is a conjugated enzyme composed of an enzymatically inactive, high molecular weight protein component (apoenzyme) and FAD (a low molecular weight, nonproteinaceous prosthetic group). Apoglucose oxidase and FAD have a high binding affinity (binding constant of around 10.sup.10 molar.sup.-1), but can be effectively separated by treatment with acidified ammonium sulfate [Swoboda, Biochim. Biophys. Acta 175: 365-379(1969)]. Swoboda demonstrated that apoglucose oxidase exists in solution mainly with the molecular configuration of a loose flexible coil, and to a lesser extent in a compact globular form, whereas when FAD is added the protein is converted to a compact, nearly spherical form having glucose oxidase activity. FAD is understood to most efficiently bind with the globular form of apoglucose oxidase to yield active glucose oxidase. Stabilization of apoglucose oxidase by intramolecular crosslinking with glutaraldehyde has been reported [Solomon et al, Biopolymers 16: 1837-1952(1977)]. As used herein, apoglucose oxidase shall be understood to include any protein preparation having apoglucose oxidase activity, i.e., having the ability to generate glucose oxidase activity upon addition of FAD, thus including unmodified apoglucose oxidase obtained by dissociation of glucose oxidase or a chemically modified form thereof.
2. Brief Description of the Prior Art
U.S. Pat. No. 4,238,565 assigned to the present assignee describes specific binding assay methods wherein FAD is employed as label and is monitored by its ability to combine with apoglucose oxidase to form active glucose oxidase. Both homogeneous and heterogeneous formats are anticipated. In a homogeneous assay for determining an antigen in a liquid medium, a test sample of the liquid medium is combined with antibody to the antigen and with a labeled conjugate comprising the antigen or an analog thereof coupled to FAD whereby any antigen from the sample competes with antigen-FAD for binding with antibody. Apoglucose oxidase is also present or is added after an appropriate incubation period and is capable of combining with antigen-FAD which has not been bound by antibody to yield active glucose oxidase. However, antibody-bound antigen-FAD is not capable of such combination with apoglucose oxidase. Consequently, the concentration of antigen in the test sample dictates the amount of measurable glucose oxidase which results. Glucose oxidase activity is measurable in a wide variety of known manners, including colorimetric methods.
The FAD-labeled homogeneous specific binding assay is generally applicable to the determination of a wide variety of ligands over a wide range of concentrations. However, application of the assay method to certain existing analytical instrumentation requiring incubation of the assay reaction mixture at elevated temperatures and for short periods has been found to be limited. It has been found that the ability of FAD and FAD-derivatives (i.e., ligand-FAD conjugates) to combine with apoglucose oxidase to form active glucose oxidase decreases rapidly with increasing temperature above room temperature, to the point that at temperatures above about 30.degree. C. significantly greater amounts of apoglucose oxidase must be added to the assay reaction mixture to obtain a dose-response curve. At 35.degree.-40.degree. C., the FAD-apoglucose oxidase recombination reaction is slowed to the point that analytically useful dose-response curves are not possible to obtain with short incubation periods (e.g., 10 minutes or less) for the determination of ligands at concentrations below 10.sup.-6 M.
The preparation of antibody to glucose oxidase is reported by Green et al, J. Immunol. 104: 1094-1100(1970). Apoglucose oxidase preparations useful in the FAD-labeled specific binding assay method are described in U.S. Pat. No. 4,268,631 assigned to the present assignee.